Hepatitis B Virus Reverse Transcriptase and {varepsilon} RNA Sequences Required for Specific Interaction In Vitro

Initiation of reverse transcription and nucleocapsid assemblyin hepatitis B virus (HBV) depends on the specific recognitionof an RNA signal (the packaging signal, ${varepsilon}$ ) on the pregenomicRNA by the viral reverse transcriptase (RT). Using an in vitroreconstitution system whereby the cellular heat shock protein90 chaperone system activates recombinant HBV RT for specific ${varepsilon}$ binding, we have defined the protein and RNA sequences requiredfor specific HBV RT- ${varepsilon}$ interaction in vitro. Our results indicatedthat approximately 150 amino acid residues from the terminalprotein domain and 230 from the RT domain were necessary andsufficient for ${varepsilon}$ binding. With respect to the ${varepsilon}$ RNA sequence,its internal bulge and, in particular, the first nucleotide(C) of the bulge were specifically required for RT binding.Sequences from the upper portion of the lower stem and the lowerportion of the upper stem also contributed to RT binding, asdid the base pairing of the upper portion and the single unpairedU residue of the upper stem. Surprisingly, the apical loop of ${varepsilon}$ , known to be required for RNA packaging, was entirely dispensablefor RT binding. A comparison of the requirements for in vitroRT- ${varepsilon}$ interaction with those for in vivo pregenomic RNA (pgRNA)packaging clearly indicated that RT- ${varepsilon}$ interaction was necessarybut not sufficient for pgRNA packaging. In addition, our resultssuggest that recognition of some ${varepsilon}$ sequences by the RT may berequired specifically for viral DNA synthesis.

INTRODUCTION

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Introduction

Hepatitis B virus (HBV) infection is a major global public healthproblem, with over 300 million chronically infected patientsworldwide (34). Chronic HBV infection carries a great risk ofdeveloping severe liver diseases, including cirrhosis and livercancer, which result in a million deaths annually (4, 10). HBVis a member of the Hepadnaviridae family, a group of small hepatotropicDNA viruses that also includes the related animal viruses, suchas the duck hepatitis B virus (DHBV) and the woodchuck hepatitisvirus. All hepadnaviruses carry a small (ca. 3.2-kb), relaxedcircular, partially double-stranded DNA genome and replicatethis DNA genome through an RNA intermediate, the pregenomicRNA (pgRNA), by reverse transcription (45, 46).

Hepadnaviruses encode a novel, multifunctional reverse transcriptase(RT). Like its retroviral counterparts, the hepadnavirus RTcatalyzes RNA- and DNA-dependent DNA polymerization and hasan intrinsic RNase H activity (12, 40, 49). Thus, the centralcatalytic RT domain and carboxyl (C)-terminal RNase H domainof the hepadnavirus RT are homologous to the corresponding domainsof retroviral RTs. However, all hepadnavirus RTs share an amino(N)-terminal domain called the terminal protein (TP) (2, 12,40), which is absent from retroviral RTs and unrelated to anyother known proteins. TP is separated from the RT domain bya nonessential and nonconserved spacer (tether) region. Theunique TP domain is used as a protein primer to initiate reversetranscription catalyzed by the conserved RT domain (54, 59,60). In addition to having this dual role as a primer and apolymerase in viral DNA synthesis, the RT is also essentialfor the packaging of the pgRNA into viral nucleocapsids (1,12, 17), the locale of reverse transcription.

Critical to both protein-primed initiation of reverse transcriptionand assembly of replication-competent nucleocapsids is the abilityof the RT to specifically recognize and form a ribonucleoprotein(RNP) complex with a short RNA signal located at the 5' endof the pgRNA, called ${varepsilon}$ (21, 39, 55). Initially identified asthe RNA packaging signal (28) that directs the specific encapsidationof the pgRNA into nucleocapsids, ${varepsilon}$ was subsequently found toalso be the origin of reverse transcription (36, 48, 53). Bindingof the RT to ${varepsilon}$ thus triggers two critical early steps in hepadnavirusreplication, the initiation of reverse transcription, via proteinpriming (14, 31, 36, 42, 53, 55), and nucleocapsid assembly,leading to the selective incorporation of both the RT and pgRNAinto the viral cores (1, 3, 39).

Detailed biochemical studies of RT- ${varepsilon}$ interaction, using the DHBVmodel system, have been made possible by the development ofcell-free systems in recent years (19, 22). The first breakthroughcame in 1992, when Wang et al. succeeded in expressing a DHBVRT active in ${varepsilon}$ binding and protein priming using a rabbit reticulocytelysate in vitro translation system (54, 55). More recently,we have been able to obtain purified recombinant DHBV RT proteinsusing the bacterial expression system and truncated mini RTconstructs that proved to be more readily expressed (20, 25,56-58). Studies using these in vitro systems, together withgenetic studies with cell cultures, have demonstrated that RT- ${varepsilon}$ interaction in DHBV requires sequences from both the TP andRT domains of the RT protein (39, 44, 55) and both sequenceand structural determinants of the ${varepsilon}$ RNA (6, 9, 39, 55). Furthermore,these studies demonstrated that the DHBV RT protein requiresthe assistance of a cellular molecular chaperone complex consistingof heat shock protein 90 (Hsp90) and several cofactors in orderto interact specifically with ${varepsilon}$ (7, 20, 23, 25, 26, 56).

The ${varepsilon}$ RNA sequences from all hepadnaviruses bear two invertedrepeats and can form a conserved stem-loop structure. The predictedstructure, which is partially supported by enzymatic and chemicalmapping studies (5, 28, 29, 38), features a lower and an upperstem, an apical loop, and an internal bulge. In vitro RNA bindingstudies, again using the DHBV system, suggest that the RT mayrecognize mainly structural features of ${varepsilon}$ , with only limiteddirect recognition of specific nucleotide sequence (6, 9, 38,39, 43; for a review, see reference 24).

In contrast to DHBV, in vitro assays to measure specific RT- ${varepsilon}$ interaction in HBV were not available until very recently; asa result, the sequence and structural determinants of the HBVRT and ${varepsilon}$ important for RNP formation have not been clearly defined.In vivo experiments in cell cultures have shown that an HBV ${varepsilon}$ stem-loop structure similar to that of the DHBV ${varepsilon}$ may also beimportant for its interaction with the HBV RT, as inferred fromthe requirements for pgRNA packaging and DNA synthesis in cells(3, 14, 28, 36). However, studies using DHBV have clearly shownthat pgRNA packaging and DNA synthesis in vivo have overlappingbut distinct requirements compared with those for RT- ${varepsilon}$ interactionin vitro (11, 13, 17, 18, 37).

In order to study the HBV RT- ${varepsilon}$ interaction directly, we haverecently developed an in vitro reconstitution system for HBVRNP formation, using purified HBV RT proteins expressed in bacteriaand components of the Hsp90 chaperone complex, similar to whatwe had done previously using the DHBV system. Using this reconstitutionsystem, we reported recently that similar to DHBV, HBV RT requiresboth its TP and RT domains for ${varepsilon}$ binding (21), but the boundariesof these domains required for ${varepsilon}$ recognition were undefined. Similarly,we reported that deletion of the internal bulge, but not theapical loop, of the HBV ${varepsilon}$ RNA abolished RT binding, but the exactsequence and structural determinants of ${varepsilon}$ recognized by the RTremained to be elucidated. To better understand the molecularbasis of specific HBV RT- ${varepsilon}$ interactions, we have now carriedout a detailed mapping study of the protein and RNA sequencerequirements for specific RT- ${varepsilon}$ interaction in HBV. A comparisonof the requirements for RT- ${varepsilon}$ interaction in vitro with thoserequired for pgRNA packaging and DNA synthesis in cells revealedthat distinct RT- ${varepsilon}$ interactions may be required specificallyfor pgRNA packaging or viral DNA synthesis, and both RNA packagingand DNA synthesis entail additional requirements beyond RNPformation.

MATERIALS AND METHODS

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Materials and Methods

Plasmids. The HBV (strain ayw) RT coding sequences were derived from pCMVHBV(14), kindly provided by Christoph Seeger. The starting andending amino acid positions of the different segments of theRT that were expressed are as indicated in Fig. 1. Deletionsand truncations were generated using appropriate restrictionenzyme digestions and are denoted by the appropriate restrictionsites. DNA coding sequences for these fragments were subclonedinto pGEX-KT (16) so that the glutathione S-transferase (GST)was fused in-frame to the N terminus of the RT sequences inthe resulting fusion proteins, as described before (21). pCI-HEwas constructed by subcloning the HBV sequence from nucleotides(nt) 1803 to 1988, encompassing direct repeat 1 (DR1) 5' of ${varepsilon}$ , ${varepsilon}$ , and 80 nt 3' of ${varepsilon}$ , into the vector pCI (Promega), downstreamof the T7 promoter.

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FIG. 1. Expression of HBV RT truncation and deletion mutants. (A) Schematic diagram of the HBV RT proteins and domains expressed. Shown on the top is the domain structure of the HBV RT, with the primer tyrosine (Y63) and the double aspartate (D540/D541) in the RT active site denoted. The cross-hatched box denotes an insertion sequence (residues 447 to 500) in the HBV RT (and other mammalian RTs) relative to the DHBV RT, based on sequence alignment. The ends of the truncations and deletions are indicated. The ${varepsilon}$ binding activities of the RT mutants are summarized to the right. The shaded bars represented in the diagram for construct HTPRT/Bsa-Spe denote the boundaries of the TP and RT domains required for ${varepsilon}$ binding in vitro. (B) The truncated HBV RT proteins and domains were expressed as GST fusion proteins in bacteria, purified using glutathione resins, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by Coomassie blue staining. The intact fusion proteins are indicated by asterisks. The bacterial chaperone protein GroEL (GL), copurifying with the RT proteins, is indicated by an arrowhead. The major degradation product, GST, is also indicated. M, molecular mass markers. Note that the first seven constructs in panel A have been reported recently (21).

Protein expression and purification. GST-tagged HBV RT fusion proteins were expressed in Escherichiacoli and purified by using the glutathione affinity resin asdescribed before (20, 21). Chaperone proteins were purifiedas previously described (21).

RNA binding. The HBV and DHBV ${varepsilon}$ RNAs were made from a synthetic DNA templateby in vitro transcription using the MegaScript kit (Ambion)as described previously (20, 21). The HE RNA was made from thepCI-HE plasmid by linearizing it immediately after the HBV sequence,followed by in vitro transcription using the T7 promoter. Tomake labeled RNAs, [ ${alpha}$ -³²P]UTP was included in the transcriptionreaction mixture. The wild-type, or "long," HBV ${varepsilon}$ RNA correspondedto that described before (24, 28). The short (shorter lowerstem), dB (bulge deletion), and dL (loop deletion) HBV ${varepsilon}$ RNAshave been described recently (21). Other deletion and substitutionmutants of ${varepsilon}$ are as described in Fig. 7. Binding of the GST-HBVRT fusion proteins to the ${varepsilon}$ RNA was measured by a reconstitutedin vitro RNA gel mobility shift assay, as described recently(21). Briefly, approximately 20 ng purified GST-RT fusion proteinswere incubated in 10-µl reaction mixtures with approximately20 ng of ³²P-labeled RNA, together with Hsp90 (360 ng), Hsp70(3 µg), Hdj1 (200 ng), Hop (370 ng), and p23 (100 ng).After incubation for 2 h at 30°C, the reaction mixtureswere resolved on 5% native polyacrylamide gels. Autoradiographyof the dried gels was then used to detect the labeled RNA andRNA-protein complexes. Where indicated, cold, unlabeled RNAswere added at 100-fold molar excess relative to the concentrationsof labeled RNAs as competitors.

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FIG. 7. Summary of RT binding activities of HBV ${varepsilon}$ RNA mutants. Left. The wild-type (long) HBV ${varepsilon}$ sequences are shown at the top, with the lower-stem, bulge, upper-stem, and loop structural elements indicated. Deletions are denoted by the dots at the appropriate positions, and substitutions are indicated by lowercase, bold, and underlined lettering. Middle. The RNA mutants are schematized in the secondary-structure model. Deletions are denoted by dotted lines and substitutions by thick lines. Right. RT binding activities of the ${varepsilon}$ RNAs are summarized. The following symbols are used to indicate the RT binding activity: ++, 50 to 100% of wild-type activity; +, 10 to 50%; +/–, <10%; –, undetectable activity. The RNA packaging activities of the various ${varepsilon}$ mutants are taken from the literature (see Discussion for details). **, DNA synthesis is blocked; ***, deduced from the RNA selection study; ?, no effect on pgRNA packaging though defective in ${varepsilon}$ binding. The different degrees of shading and the numerals 1 to 4 are used to denote the classification of the ${varepsilon}$ mutants into four different groups (see Discussion for details).

RESULTS

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Results

Mapping of RT sequences required for ${varepsilon}$ binding. We have recently reported that both the TP and RT domains ofthe HBV RT protein were required for specific binding to thecognate HBV ${varepsilon}$ RNA, as determined by using an in vitro RNA mobilityget shift assay (21) that can directly measure specific HBVRT- ${varepsilon}$ interaction (for details, see Materials and Methods). Usingthis assay, we have carried out a detailed mapping of the sequenceswithin the TP and RT domain that are required for ${varepsilon}$ binding.As depicted in Fig. 1, truncations were made from both the Nand C termini, as were deletions from within the spacer region.The truncated HBV RT proteins were expressed and purified asGST fusion proteins at similar levels (Fig. 1B and data notshown). The ability of the purified RT proteins to bind ${varepsilon}$ wasdetermined by incubating them with radiolabeled HBV ${varepsilon}$ RNA andfive purified Hsp90 chaperone components, i.e., Hsp90, Hsp70,Hop (p60), Hdj-1, and p23, as described before (21). These studiesshowed that the TP domain sequences from residues 42 to 196,together with the RT domain sequences from residues 291 to 520,as represented by the construct HTPRT/Bsa-Spe, were sufficientfor specific ${varepsilon}$ binding (Fig. 1 and 2). Thus, the spacer sequencesfrom at least residues 197 to 290, the entire RNase H domain,at least 41 residues from the N-terminal portion of the TP domain,and the C-terminal portion of the RT domain, including the YMDDcatalytic motif, were dispensable for ${varepsilon}$ binding, indicating that ${varepsilon}$ binding activity can be genetically separated from any knownenzymatic activities of the RT protein.

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FIG. 2. In vitro ${varepsilon}$ RNA binding activity of HBV RT mutants as detected by the gel mobility shift assay. Purified HBV RT proteins, all as GST fusion proteins, were incubated with ³²P-labeled HBV ${varepsilon}$ RNA in the presence of the Hsp90 chaperone factors as detailed in Materials and Methods. The binding reaction mixtures were resolved on a native polyacrylamide gel, which was then dried and subjected to autoradiography. The labeled free ${varepsilon}$ RNA and the RT- ${varepsilon}$ complex are indicated.

Mapping of ${varepsilon}$ sequences required for RT binding. The HBV ${varepsilon}$ RNA is thought to adopt a secondary structure, witha lower and an upper stem, an internal bulge, and an apicalloop, which is depicted schematically in Fig. 3 to 8. To dissectthe contribution of each of the ${varepsilon}$ structural elements to specificRT binding, we carried out a systematic-mutagenesis study witheach of these structural elements. To help correlate the invitro RT binding activity of the ${varepsilon}$ RNA mutants with their invivo pgRNA packaging function, we chose to construct most ofthe ${varepsilon}$ RNA mutants corresponding to those previously tested inthe pgRNA packaging assay (see Discussion below). To ascertainwhether the different RT proteins would bind the different ${varepsilon}$ RNA mutants differentially, each ${varepsilon}$ mutant was assayed for bindingto multiple RT proteins. However, all RT proteins tested bounda given RNA similarly (e.g., Fig. 3), and only the results obtainedwith one particular RT protein for any given ${varepsilon}$ mutant is thusshown below.

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FIG. 3. In vitro binding of HBV RT proteins to ${varepsilon}$ RNA mutants with the apical loop but not the internal bulge deleted. Two different RT proteins were tested for their binding to the wild-type (long), lower-stem-shortened (short), bulge deletion (dB), or loop deletion (dL) ${varepsilon}$ RNA, as described in the legend to Fig. 2. The RNA mutants are schematized in the secondary-structure model. Deletions are denoted by dotted lines. The symbol * indicates a minor structural isoform detected sometimes in the free RNA, as reported before (21).

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FIG. 8. Model of HBV ${varepsilon}$ binding by the RT and putative host factor. The HBV ${varepsilon}$ RNA is depicted as the typical stem-loop structure. Critical sequences for RT binding (bounded by the dotted line) center around the internal bulge, including the first two nucleotides of the bulge, the upper portion of the lower stem, and the lower portion of the upper stem. The apical loop, dispensable for RT binding but critical for pgRNA packaging, may interact with an unknown host factor(s) (HF) in directing pgRNA packaging and possibly protein priming. See the text for detailed discussions of the model.

Lower stem. Since we have previously shown that the lower portion of thelower stem was dispensable for RT binding in the DHBV system(20), we were interested in determining if this was also thecase for the HBV ${varepsilon}$ RNA. As depicted in Fig. 3, 4, and 7, removalof the bottom seven base pairs from the lower stem of the HBV ${varepsilon}$ did not significantly impair the ability of the deletion mutant(short RNA) to bind the RT, as determined by both a direct bindingassay, where the mutant ${varepsilon}$ was labeled as the probe (Fig. 3 and4B), and a competition assay, where the mutant ${varepsilon}$ was added asa competitor in a binding reaction mixture with the labeled,wild-type (long) ${varepsilon}$ serving as the probe (Fig. 4A). In addition,we tested a longer HBV RNA, called HE (Fig. 4), encompassingnot only the ${varepsilon}$ but also the DR1 sequence upstream and 80 nt downstream.This longer RNA did not compete any better than the short HBV ${varepsilon}$ RNA in the competition assay, indicating that the short HBV ${varepsilon}$ RNA contained all the essential sequences for RT binding invitro. Thereafter, we chose to construct most of the ${varepsilon}$ mutationsin the shorter ${varepsilon}$ RNA background for convenience.

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FIG. 4. Binding of HBV RT to ${varepsilon}$ mutants. (A) HTPRT/Drd was incubated with ³²P-labeled wild-type HBV ${varepsilon}$ RNA, as described for Fig. 2, in the presence of the indicated unlabeled RNA competitors (Compet). The RNA mutants are schematized in the secondary-structure model. Deletions are denoted by dotted lines and substitutions by thick lines. Refer to Fig. 7 for the exact deletions/substitutions in each of the ${varepsilon}$ mutants tested. HE is a 190-nt-long HBV RNA, encompassing DR1 5' of ${varepsilon}$ , ${varepsilon}$ , and 80 nt 3' of ${varepsilon}$ (see Materials and Methods for details). The DHBV ${varepsilon}$ and tRNA, which cannot bind to the HBV RT (21), were used as nonspecific competitors. All competitor RNAs were used at a 100-fold molar excess. Note that all reactions also contained an additional 300-fold molar excess of tRNA over the labeled probe (21). (B) HTPRT/Drd (lanes 1 to 7) or HTPRT/Spe (lanes 8 to 11) was tested for binding to the ³²P-labeled HBV ${varepsilon}$ RNA mutants, as described for Fig. 2.

To test the role of the upper portion of the lower stem in RTbinding, we changed the nucleotide sequence but maintained thebase pairing in this region. The resulting ${varepsilon}$ mutant (L-L/R) wasseverely impaired in its ability to bind the RT in both thedirect binding and the competition assays (Fig. 4 and 7), indicatingthat base pairing here alone was not sufficient for RT bindingand that some specific sequences in the lower stem contributedto the RT interaction.

Internal bulge. We have recently reported that deletion of the entire bulgeof the HBV ${varepsilon}$ RNA (dB ${varepsilon}$ ) abolished RT binding (21) (Fig. 3 and7). To determine if the internal bulge acted only as a structuralelement or if its specific nucleotide sequence was requiredfor RT binding, we made selected base substitutions in thisregion, using mainly mutants that had previously been testedin the pgRNA packaging assay. The mutant B1-4 had the first(5') 4 nucleotides of the bulge replaced and completely lostthe ability to bind the RT (Fig. 4 and 7), indicating that atleast some of the bulge sequences were specifically requiredfor RT binding. To further ascertain the base-specific contributionof the bulge to RT binding, further substitutions were madein the bulge region. Changes atthe third and fifth positionsof the bulge (B35) did not affect RT binding at all, while changesat positions 2, 3, 4, and 6 (B2346) significantly decreasedRT binding (Fig. 5, 6B, and 7). Thus, while the B2346 mutantstill displayed specific RT binding activity, in competitionexperiments, it was less effective in competing with eitherthe short ${varepsilon}$ or the B35 mutant for RT binding. Another bulge mutant,ApaB, having substitutions at positions 1, 2, 4, and 5 lostRT binding activity completely (Fig. 6B and 7). These resultssuggested that the first two nucleotides, particularly the 5'-mostnucleotide of the bulge, may be critical for RT binding. Totest this further, we changed the first position of the bulgefrom C to all three other nucleotides. In support of our hypothesis,the single nucleotide substitution from C to G (B1G) or to A(B1A) (both transversions) completely abolished RT binding,while the less drastic transition mutation (B1U) retained alow residual RT binding activity (Fig. 6A and 7).

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FIG. 5. Binding of HBV RT to ${varepsilon}$ bulge mutants. HTPRT/Drd was tested for binding to the ³²P-labeled short (lanes 1 to 4), B2346 (lanes 5 to 8), or B35 (lanes 9 to 12) ${varepsilon}$ RNA, as described for Fig. 2. Where indicated, unlabeled RNAs (in 100-fold molar excess of the labeled RNAs) were added to the binding reaction mixtures as competitors (Compet). The RNA mutants are schematized in the secondary-structure model. Substitutions are denoted by thick lines. Refer to Fig. 7 for the exact bulge substitutions.

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FIG. 6. Binding of HBV RT to ${varepsilon}$ mutants. (A) HTPRT/Spe was tested for binding to the indicated ³²P-labeled HBV ${varepsilon}$ RNAs, as described for Fig. 2. (B) HTPRT/Drd (lanes 1 to 4) or HTPRT/Bsa-Spe (lanes 5 to 10) was incubated with the ³²P-labeled HBV short ${varepsilon}$ RNA, as described for Fig. 2. Where indicated, unlabeled RNAs (in 100-fold molar excess of the labeled RNAs) were added to the binding reaction mixtures as competitors (Compet). The RNA mutants are schematized in the secondary-structure model. Deletions are denoted by dotted lines and substitutions by thick lines. Refer to Fig. 7 for the exact ${varepsilon}$ mutations.

Upper stem. We made substitutions to the lower and upper portions of theupper stem separately due to the interesting differential requirementfor these two regions of the HBV ${varepsilon}$ RNA in pgRNA packaging (14,38). For the lower portion, we replaced three nucleotides oneither side of the stem. Interestingly, the left-side substitution(U-L-L) decreased RT binding more severely than the right-sidesubstitution (U-R-L), but both mutants retained some RT bindingactivity (Fig. 4 and 7) as measured by both direct binding andcompetition assays, despite the disruption of base pairing.Restoration of base pairing but not the sequence in the thirdmutant (U-L/R-L) did not restore RT binding completely either.These results indicated that base pairing in this region of ${varepsilon}$ was neither essential nor sufficient for RT binding and, furthermore,that the RT recognized the sequences on either side of the stemdifferentially. In sharp contrast, we found that base pairingin the upper portion of the upper stem was required and sufficientfor RT binding. Both sides of the stem could be replaced withoutaffecting RT binding as long as base pairing was maintained(U-L/R-U), while disruption of base pairing led to a completeloss of RT binding (U-L-U, U-R-U) (Fig. 4 and 7), indicatingthat the RT recognized this portion of the upper stem as a double-strandedstructural element rather than any specific sequences. Finally,deletion of the single unpaired U in the upper stem also severelydiminished RT binding (Fig. 6A and 7).

Apical loop. We have reported recently that the apical loop of the HBV ${varepsilon}$ RNAcould be deleted completely (dL), in the context of the long ${varepsilon}$ RNA, without affecting RT binding (21). We made here the samedeletion (short-dL) in the short (i.e., shortened lower stem) ${varepsilon}$ , and the resulting mutant still retained RT binding activity(Fig. 4 and 7). To confirm these results, we tested the bindingof the ${varepsilon}$ loop deletion mutant to a number of different RT truncations,and it could bind to all the RT proteins that were competentfor binding to the wild-type ${varepsilon}$ (Fig. 3 and data not shown). Ithas been shown previously that deletion of the loop leads toan altered RNA structure that nevertheless maintains the originallower stem and bulge sequence but with a shortened upper stemand a new apical loop with a completely different sequence (29),indicating again that the wild-type loop sequences are not importantfor RT binding. To further assess the role of the apical loopof ${varepsilon}$ in RT binding, we made the mutant L1-4, which has the first4 nucleotides of the loop changed and which had previously beenshown to abolish pgRNA packaging (38). Again, these substitutionsdid not affect RT binding at all (Fig. 4). Together, these resultsindicated that the apical loop of the HBV ${varepsilon}$ did not play a significantrole in RT binding.

Finally, we tested whether the lower stem and the bulge sequencesalone (Fig. 6 and 7), in the absence of the upper stem and loopsequences, would be sufficient for RT binding, in light of thedispensability of the apical loop and the only limited requirementof the upper stem. However, our results showed that the lowerstem plus the internal bulge alone were clearly insufficientfor RT binding.

DISCUSSION

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Discussion

Taking advantage of our recently established in vitro reconstitutionsystem for specific HBV RT- ${varepsilon}$ interaction, we have carried outa detailed mapping analysis of the RT and ${varepsilon}$ determinants forspecific RNP formation in vitro. The identification of thesedeterminants, together with those mapped previously for pgRNApackaging and DNA synthesis, now provides the opportunity tocritically evaluate the role of the HBV RT- ${varepsilon}$ interaction in pgRNApackaging and DNA synthesis.

With regard to the RT, our results indicate that less than 400residues from the TP domain plus the N-terminal portion of theRT domain are sufficient for specific ${varepsilon}$ binding. The spacer region,the entire RNase H domain, and the C-terminal part ofthe RTdomain, including the catalytic YMDD motif (and thusthe RT enzymaticactivity), were dispensable for ${varepsilon}$ binding. These results arein good agreement with the previously mapped sequences in theDHBV RT protein that are required for binding to its cognateDHBV ${varepsilon}$ RNA (20, 39, 44, 55). Since pgRNA packaging requires essentiallythe entire length of the RT sequence, in both HBV and DHBV (1,17), binding of the RT to ${varepsilon}$ is not sufficient for pgRNA packagingin vivo for these viruses. The C-terminal RT sequences, includingthe RNase H and part of the RT domain, which are required forpgRNA packaging but dispensable for ${varepsilon}$ binding, may mediate pgRNApackaging by interacting with the assembling core protein subunits,as reported before (35). However, since the RT alone, in theabsence of ${varepsilon}$ , cannot be packaged into nucleocapsids (3), theputative RT-core interaction is apparently productive only forRT packaging in the context of the RT- ${varepsilon}$ RNP complex. Interestingly,our results revealed that an "insert" sequence in the HBV RTdomain (residues 447 to 500) (Fig. 1) (40), which is conservedin the mammalian hepadnaviruses but absent in the DHBV RT, mayplay a critical role in ${varepsilon}$ binding.

Recombinant HBV RT proteins have previously been purified froman insect cell expression system and displayed low levels ofprotein-priming activity in vitro, although this low primingactivity could occur independently of the ${varepsilon}$ RNA (30-32, 52).The TP domain sequence (residues 20 to 199) required for thispriming activity is similar to that required for ${varepsilon}$ binding mappedhere (residues 42 to 196), as are the boundaries for the dispensablespacer region in both assays (residues 200 to 300 in the primingassay versus residues 197 to 290 for ${varepsilon}$ binding). However, proteinpriming required a much longer sequence from the C-terminalregion of the RT, including the entire RT domain and part ofthe RNase H domain as well, indicating that protein primingrequires much more RT sequence than does ${varepsilon}$ binding.

With regard to the ${varepsilon}$ RNA sequences required for RT binding, ouranalyses revealed that the main sequence determinants recognizedby the RT are (i) the internal bulge, specifically its firstand, to a lesser extent, second nucleotides, and (ii) the sequencessurrounding the bulge, including the lower part of the upperstem (particularly its left-side sequence) and the upper partof the lower stem, as well as the base pairing of the upperpart and the single unpaired base of the upper stem (as summarizedin Fig. 7 and 8). A comparison of these requirements with thosemapped previously for pgRNA packaging in vivo allowed the classificationof the ${varepsilon}$ mutants analyzed here into four groups. The first group,including mutants with the shortened lower stem (short), withsome substitutions at the internal bulge (B35), and with thesubstitutions (but with maintenance of base pairing) at theupper part of the upper stem (U-L/R-U), are fully functionalin both RT binding in vitro and pgRNA packaging in vivo. Inaddition, mutants with other substitutions at the bulge (B2346)retained significant, though decreased, activity in both RTbinding and pgRNA packaging. The second group, including mutantswith a deletion of the internal bulge (dB), certain substitutionsat the bulge (ApaB, involving positions 1, 2, 4, and 5), particularlysingle substitutions at the first position of the bulge (B1G,B1A, and, to a lesser extent, B1U), disruption of the base pairingat the upper part of the upper stem (U-L-U and U-R-U), and deletionof the upper stem plus the loop (bulge), are defective in bothRT binding and pgRNA packaging. In addition, substitutions atthe lower stem (L-L/R) also severely decreased both of theseactivities. These first two groups of ${varepsilon}$ mutants underscore thecritical role of RT- ${varepsilon}$ interaction, particularly the beginningsequences of the bulge, in pgRNA packaging. The third group,including mutants with deletions (dL and short dL) and substitutions(L1-4) of the apical loop, substitutions at the right side ofthe lower part of the upper stem (U-R-L and U-L/R-L), and adeletion of the single unpaired U residue from the upper stem,are fully or partially active in RT binding but defective inRNA packaging, again indicating that RT- ${varepsilon}$ interaction is notsufficient for pgRNA packaging. The last group, including mutantswith substitutions at the left side of the lower part of theupper stem (U-L-L) and the B1-4 bulge substitution, are defectivein RT binding but retain wild-type-like ability in RNA packaging.The identification of this group of ${varepsilon}$ mutants may suggest thatsome aspects of RT- ${varepsilon}$ interaction are actually not required forpgRNA packaging but may instead be involved in another stage(s)of viral replication, e.g., DNA synthesis (see below for morediscussion on this).

Our result showing a sequence-specific contribution of the lowerstem to RT binding agrees well with previous reports implicatingboth structural and sequence-specific determinants of the lowerstem of the HBV ${varepsilon}$ RNA in pgRNA packaging (14, 38, 50, 51). Also,a DHBV-HBV hybrid ${varepsilon}$ RNA, with the HBV lower stem sequence substitutingfor the corresponding DHBV sequence, was inactive in eitherDHBV RT binding or protein priming, indicating that at leastsome specific sequences of the DHBV ${varepsilon}$ lower stem are also requiredfor DHBV RT binding (55). The retention of the RT binding activityof the HBV ${varepsilon}$ mutant with a shortened lower stem is consistentwith the dispensability of the beginning portion of the lower-stempgRNA packaging in vivo (50). We (20) and others (8) also showedthat the bottom portion of the lower stem of the DHBV ${varepsilon}$ couldbe removed without affecting DHBV RT binding or protein primingin vitro.

Our studies have revealed a critical role of the internal bulgesequence in RT binding. Although it was initially thought thatthe bulge played mainly a structural role in pgRNA packagingin that certain bulge substitutions still allowed efficientpgRNA packaging (14, 29, 38, 50), some specific bulge sequencesalso appeared to be required for pgRNA packaging. In particular,the first two nucleotides (CU) of the bulge were strongly selectedfor in a reiterative selection procedure for packaging competent ${varepsilon}$ RNA variants (41). In nature, the entire bulge sequence ishighly conserved (15, 33, 38, 41). The only naturally occurringvariation reported at the first two positions is the less drasticC-U transition at the first position (33), which we have shownhere to retain some residual RT binding activity and which wasalso selected for, at a lower frequency, in the aforementionedselection study. The conservation of the 3' four nucleotidesof the bulge underscores their role as the template for viralDNA synthesis during protein priming (14, 36). Our results showinga base-specific recognition of the 5' two nucleotides of thebulge by the RT now provides an explanation for the sequenceconservation at these two positions, which, by virtue of theirspecific interaction with the RT, play a critical role in pgRNApackaging. Surprisingly, a substitution of the first four nucleotidesof the bulge (B1-4) was previously reported to be packagingcompetent (38), although the same mutant was shown here to becompletely defective in RT binding. We note that the loss ofRT binding by the B1-4 mutant is consistent with the deleteriouseffect of substitutions at the first and second positions ofthe bulge on RT binding that we observed here in vitro and thestrong selection previously reported for the wild-type sequencesat these same positions in the RNA packaging assay (41).

The lower portion of the upper stem has been shown to contributeto pgRNA packaging. In particular, the specific nucleotide sequenceat the right side seemed to be critical (38), and restorationof base pairing (but not sequence) could not restore pgRNA packaging(38, 51). The same right-side substitution mutant shown to bedefective in pgRNA packaging (38) also displayed decreased RTbinding activity, and base pairing here alone also was not sufficientto restore RT binding, indicating that nucleotide-specific recognitionof the right side by the RT plays an important role in HBV pgRNApackaging. However, replacement of the left-side sequences herecaused an even more severe defect than the right-side substitutionin RT binding, although the same left-side substitution mutantwas active in pgRNA packaging (38). Intriguingly, other left-sidesubstitutions also showed no defect in pgRNA packaging but insteadblocked viral DNA synthesis (14), suggesting that nucleotide-specificrecognition of these ${varepsilon}$ sequences by the RT, as detected herein our in vitro binding assay, may play an important role insome aspects of viral reverse transcription. For example, theRT may facilitate the proposed long-range interaction of thisregion of the ${varepsilon}$ with the sequences near the 3' DR1 to facilitatetemplate switching from ${varepsilon}$ to DR1 during minus-strand DNA synthesis(47; D.Loeb, personal communication). Our analyses have, thus,begun to reveal that distinct RT- ${varepsilon}$ interactions may be involvedin viral DNA synthesis as opposed to RNA packaging. The differentialrequirements of the two sides of the lower portion of the upperstem, the insufficiency of base pairing alone, and the retentionof partial activity without base pairing for RT binding, pgRNApackaging, and DNA synthesis all suggest that this portion ofthe upper stem contributes to these functions in both structure-and sequence-specific ways, and it may in fact be at least partiallymolten upon RT binding in the RNP complex.

In contrast, the upper portion of the upper stem seems to playmainly a structural role in RT binding and pgRNA packaging.Thus, we have shown here that base pairing, but not the specificsequence, in this region was required for RT binding, and othershave shown this also to be the case for RNA packaging (38).In addition, our results indicate that recognition of the single,unpaired U bulge in the upper stem, which is conserved in allmammalian HBVs, by the RT plays an important role in pgRNA packaging,as its deletion led to defects in both RT binding and pgRNApackaging (38, 51).

The most intriguing result in our ${varepsilon}$ mapping studies was the findingthat the apical loop was entirely dispensable for RT binding.Deletion of the entire 6-nt loop or simultaneous substitutionsat the first four positions had no effect on RT binding. Previousmutagenesis studies have shown that the loop is absolutely requiredin vivo for RNA packaging (29, 38, 51), in particular, thisregion could tolerate few substitutions without causing a defectin RNA packaging. These results have led to the suggestion thatthe RT protein may make sequence-specific contacts with theloop in directing pgRNA packaging. A recent nuclear magneticresonance structure of the HBV ${varepsilon}$ RNA derivative, containing onlythe upper stem and the apical loop, suggests that the apicalloop actually consists of only 3 nucleotides (15), rather than6 as originally proposed. Regardless, it is clear that theseloop sequences could be deleted or replaced without affectingRT binding. We suggest that the requirement for the apical loopin pgRNA packaging does not result from its direct recognitionby the RT but rather from its interaction with some yet-to-be-identifiedcellular factor, both of which, together with the viral RT,are required for RNA packaging (Fig. 8).

It is interesting to contrast the dispensability of the HBV ${varepsilon}$ loop for HBV RT binding with the sequence-specific recognitionof the DHBV ${varepsilon}$ loop by its cognate DHBV RT (6, 9). On the otherhand, while base pairing of the upper stem near the HBV ${varepsilon}$ loopis required for HBV RT binding, base pairing in the corresponding ${varepsilon}$ region in the avian viruses is not required for RT binding(27, 55). Also, the specific sequences at the beginning of theDHBV ${varepsilon}$ internal bulge do not seem to be important for DHBV RTbinding, in contrast to the critical role of the analogous sequencesof the HBV ${varepsilon}$ in HBV RT binding observed here. It is possiblethat some of the differences observed may be a reflection ofthe inherent difficulty in predicting RNA structures and potentiallyunanticipated structural alterations of the ${varepsilon}$ RNA by some ofthe mutations. There also seemed to be some structural flexibilityin the ${varepsilon}$ RNA that was compatible with RT binding; either theRT could recognize multiple RNA structures or, upon binding,a common ${varepsilon}$ structure could be induced from the different protein-freeRNA structures (6, 9). Clearly, a full understanding of themolecular basis for the specific interactions between the RTand ${varepsilon}$ RNA in hepadnaviruses awaits high-resolution structuralstudies. Nevertheless, biochemical and mutational studies havehelped to define the basic requirements of both the RT proteinand the ${varepsilon}$ RNA. They also make it likely that significant differencesexist in the interactions between the RT protein and its cognate ${varepsilon}$ RNA in the different hepadnaviruses. These differences in RT- ${varepsilon}$ interactions might be in part responsible for some of the disparitiesobserved with respect to pgRNA packaging and protein primingbetween these related viruses, which still elude explanations.For example, the DHBV, but not HBV, pgRNA requires a secondsignal for packaging (11, 18, 24, 37), and the DHBV, but notHBV, RT can carry out ${varepsilon}$ -dependent protein priming under almostidentical in vitro conditions (21, 25).

ACKNOWLEDGMENTS

We thank David Toft for providing the chaperone proteins, DafnaFlores for technical assistance, and Dan Loeb for a criticalreading of the manuscript.

This work was supported by a Public Health Service grant, R01AI43453, from the National Institutes of Health.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, H107, The Penn State University College of Medicine, 500 University Dr., Hershey, PA 17033. Phone: (717) 531-6523. Fax: (717) 531-6522. E-mail: juh13{at}psu.edu.

REFERENCES

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